Every year, millions of people within the U.S. suffer from conditions like epilepsy, stroke, traumatic brain injury, Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease while billions of dollars are spent treating them in our healthcare system. All of these conditions have in common the presence of excitotoxic injury in the brain, which is characterized by increased neuronal activity and cell death. We are studying a form of adaptive synaptic plasticity that may be neuroprotective during such excitatory insults in addition to its role for setting synaptic efficacy during normal activity. In glutamatergic neurons of the hippocampus, the number of functionally active presynaptic terminals is decreased after prolonged periods of increased activity. In addition to neuroprotection, we believe that this activity-dependent plasticity may underlie temporary learning and memory problems that follow acute brain insults due to the subsequently depressed synaptic activity and may also be vital for neuronal development and network maintenance. This proposal aims to elucidate the cellular mechanisms responsible for inducing adaptive presynaptic silencing by utilizing sophisticated imaging, electrophysiological, and molecular biological techniques in primary rat hippocampal culture. The goal of Aim 1 is to determine whether adaptive presynaptic silencing is a cell- autonomous process or whether signals passed between cells are necessary for induction. We will do so by depolarizing individual neurons within a network of non-depolarized cells and by testing the role of postsynaptic neurons and glial cells. This will clarify the source of the signaling cascade responsible for this synaptic phenomenon and will contribute to our currently limited understanding of how neurons adjust to their dynamic extracellular environments. We have preliminary data that implicate inhibitory G-proteins in the induction of adaptive presynaptic silencing, so the goal of Aim 2 is to begin elucidating the upstream and downstream G-protein signaling cascades. We will test whether G-protein-coupled receptors upstream of inhibitory G-proteins modulate silencing and whether cAMP signaling is depressed during prolonged periods of increased activity. This aim identifies potential pharmacological targets to exploit for treatment of excitotoxic injury or its reversible side effects. Overall, this project will elucidate upstream signaling mechanisms governing an underappreciated form of synaptic malleability and will provide training for the applicant in techniques important for a future career in cellular neuroscience.